The expression and function of Frizzled-7 in human renal cell carcinoma

Purpose

Wnt/β-catenin has emerged as an important signal pathway in renal cell carcinoma (RCC) pathogenesis. Frizzled 7 (Fzd7) is a member of Frizzled (Fzd) receptor family which binds with Wnt ligands and transduces canonical and non-canonical pathways. However, the expression of Fzd7 in human RCC is poorly investigated.

Methods

53 RCC tissues and peri-tumor tissues were collected from the patients treated with radical nephrectomy. The expression of Fzd7 was investigated by immunohistochemical staining. Three RCC cells were transfected with Fzd7shRNA and GFPshRNA to investigate the function of Fzd7 in RCC cells.

Results

The immunohistochemical analysis showed that Fzd7 protein expression level was significantly increased in RCC tissues when compared with peri-tumor tissues, which suggested that Fzd7 might be involved in the formation of tumors. However, the Fzd7 expression was not correlated with clinicopathological parameters. Three RCC cell lines: 786-O, Caki-1, and OS-RC-2 also expressed Fzd7. With Fzd7 expression being interfered by shRNA, the RCC cell proliferation was mildly decreased. Wnt3a could stimulate the RCC cells proliferation, but the stimulation was decreased when Fzd7 expression was interfered. Restoring the Fzd7 expression led to the proliferation stimulation effect of Wnt3a being restored.

Conclusions

This paper suggests that Fzd7 may act as one of the molecules that take part in the course of RCC formation. Fzd7 can be activated by Wnt3a to stimulate cell proliferation.

     

围手术期感染对肾细胞癌切除术后肿瘤特异性生存期的影响

目的 探讨围手术期感染对肾细胞癌切除术后肿瘤特异性生存期(CSS)的影响。方法 以青岛大学附属医院2006年1月—2013年1月收治的1 800例肾癌患者为研究对象,分析肾癌切除术后30 d内发生感染的情况。用Kaplan-Meier曲线分析感染与未感染患者的CSS;建立Cox比例风险模型来评估感染对CSS的影响时控制性别、年龄、AJCC分期和分级、肿瘤大小、病理学类型、合并症、手术方式和系统治疗等因素。结果 1 800例患者中位年龄74岁(69~79岁),中位随访时间为42个月(22~67月)。严重感染者141例,非严重感染者296例,未感染者1 363例。Kaplan-Meier分析结果显示,严重感染有改善肾癌(肿瘤直径为7.0~9.9 cm)患者CSS趋势(P=0.048);对于肿瘤直径≥10 cm或＜7 cm的患者,严重感染对CSS无影响(0.1~3.9 cm,P=0.120;4.0~6.9 cm,P=0.768;≥10 cm,P=0.412)。多因素Cox回归分析结果显示,严重感染可以改善肾癌患者的CSS(HR=0.752,P=0.032)。这种效应在肿瘤直径≥7 cm的患者中较为明显(P=0.041),而在肿瘤直径＜7 cm的患者中表现得不明显(P=0.268)。结论 围手术期严重感染可以改善肿瘤直径7~10 cm的肾癌患者CSS,但是对肿瘤直径≥10 cm或＜7 cm肾癌患者的CSS无明显影响。     

Lack of interleukin-2 (IL-2) expression and selective expression of IL-10 mRNA in human renal cell carcinoma

Freshly isolated tumor-infiltrating lymphocytes (TIL) are often functionally deficient. Since one of the key functional parameters of an immune response is the local production of cytokines, we studied the expression of cytokine genes in freshly isolated renal cancer tissue. Using a PCR-assisted mRNA amplification assay, the constitutive expression of mRNA for 10 different cytokines was assessed in renal cancer tissue. We compared the cytokine mRNA expression in freshly isolated samples of renal carcinomas, renal cancer cell lines established from the tumor samples, peripheral blood mononuclear cells (PBMC) and non-tumor kidney tissue isolated from the same patients. IL-10 mRNA expression was detected only in tumor samples, while renal cancer lines, PBMC and non-tumorous kidney tissues were devoid of this cytokine. One-third of the tumor samples but none of the normal kidney samples also expressed G-CSF mRNA. IL-6, TNF-α and IFN-γ mRNA were expressed non-selectively in tumors, PBMC and normal renal tissue. Expression of IL-2, IL-3 and IL-4 mRNA was not detected in any of the tissues analyzed. Established renal cancer lines exhibited expression of IL-lα, IL-6, TNF-α and GM-CSF. Culture of tumor-derived T cells with anti-CD3 monoclonal antibody (MAb) resulted in expression of IL-2, IL-3 and IL-4 mRNA. In contrast, none of these cytokines was detected in culture with recombinant human IL-2 alone. Since IL-10 is known to suppress antigen presentation, these findings have important implications for the possible in vivo role of IL-10 as a suppressor of local anti-tumor response.     

Expression of matrix metalloproteinases 7 and 2 in human renal cell carcinoma

Matrix metalloproteinases (MMPs) are associated with invasion and metastasis of several malignant tumors in human. Especially so MMP-7, as it is mainly produced by the cancer cell itself. However, the expression of MMP-7 in renal cell carcinoma (RCC) has not been investigated. We examined expressions of MMP-7 and MMP-2 in RCC, and compared them with the clinicopathological characteristics in RCC. Tumor samples from 20 patients with RCC, who had surgery performed at Osaka City University Medical School Hospital, were immunohistochemically stained. Expression of MMP-2 was significantly stronger in advanced stage and in high grade tumors than in low stage and low grade tumors. MMP-7 was also expressed in RCCs, with its expression especially and significantly stronger in high grade than in low grade tumors (p=0.004). However, there was no significant difference of MMP-7 expression between advanced and low stages (p=0.1859). This increased expression of MMP-7 in high grade RCC might be associated with tumor invasion and metastasis.     

Prognostic factors for survival in patients with advanced renal cell carcinoma treated with recombinant interleukin-2.

A database of 327 patients with advanced Renal Cell Carcinoma (RCC) has been analyzed in order to identify potential baseline prognostic factors predicting for survival, following recombinant Interleukin-2 treatment (rIL-2). All patients received a continuous infusion (CIV). Eligibility criteria were uniform across studies, and included patients with an ambulatory performance status (PS), measurable disease, no CNS metastases, and no major organ compromise. Multivariate analyses identified baseline PS (ECOG 0 vs. 1), time from diagnosis to treatment (DTI greater than 24 months vs. less than or equal to 24 months), and the number of metastatic sites (1 vs. greater than or equal to 2, where lung, bone and other sites are considered as separate sites) as important predictors for survival. Patients can be classified into 4 subgroups, which are a function of the number of risk factors present. Median survival for each subgroup is 28, 17, 10 and 5 months, respectively. The model was validated in an independent cohort of 125 patients with RCC treated with subcutaneous (s/c) rIL-2, and predicted for survival accurately. By determining in which risk group category patients may fall, treating physicians may be better equipped to decide on patient management. The model may also be of value in order to stratify patients in randomized clinical trials.     

Significance of COX-2 expression in human renal cell carcinoma cell lines

Accumulating evidences indicate that cyclooxygenase (COX)-2 plays an important role in tumorigenesis in many human cancers. Yet the relationship between COX-2 and human renal cell carcinoma (RCC) remains unclear. The aim of our study was to evaluate COX-2 expression in human RCC cell lines and its role in tumorigenesis of human RCC. Among the human RCC cell lines (SMKT-R4, OS-RC-2, ACHN) and normal renal cell line RPTEC, COX-2 overexpression was found in OS-RC-2 cells both at mRNA and protein levels. COX-2 sense- and antisense-orientated vectors were constructed and transferred into RCC cells. Significant suppression of cellular proliferation was demonstrated in OS-RC-2 antisense transfectants, whereas promotion was found in SMKT-R4 sense transfectants by colony-forming assay despite the observation that COX-2 specific inhibitor NS398 exhibited similar IC50 among RCC cell lines by MTT assay. In comparison with parent cells and sense transfectants, significant suppression of COX-2 expression and PGE2 production and increase in butyrate-induced apoptosis were observed in OS-RC-2 antisense transfectants by Western blot, ELISA assay and FACS analysis, respectively. Furthermore, tumor growth and angiogenesis of OS-RC-2 antisense transfectants in nude mice was significantly suppressed and the survival time of these mice was significantly prolonged. Our study demonstrates that COX-2 is overexpressed in OS-RC-2 RCC cell line and plays an important role in tumorigenesis of the cells in vivo, which implies that COX-2 may be a therapeutic target for COX-2-expressing RCC, and that suppression of COX-2 expression by antisense-based strategy may have potential utility in treatment of COX-2-expressing RCC.     

Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma

PURPOSE: Although the prognostic value of B7-H1 and B7-H4 expression by tumor cells in clear cell renal cell carcinoma (ccRCC) has been established, the role of B7-H3 is unknown. As such, we evaluated the association of B7-H3 expression with clinicopathologic outcomes in patients treated for ccRCC. EXPERIMENTAL DESIGN: Nephrectomy specimens from 743 consecutive patients treated for ccRCC at our institution from 1990 to 1999 were evaluated for B7-H3 expression by immunohistochemical staining. Associations of B7-H3 expression with clinical and pathologic features were evaluated using chi2 and Fisher's exact tests. Associations of B7-H3 expression with death from RCC were evaluated using Cox proportional hazards regression models. RESULTS: B7-H3 expression by tumor cells or tumor vasculature was noted in 17% and 95% of specimens, respectively. The presence of either tumor cell or diffuse tumor vasculature expression of B7-H3 was present in 46% of specimens and was associated with multiple adverse clinical and pathologic features. After multivariable adjustment, the presence of either tumor cell or diffuse tumor vasculature B7-H3 expression was significantly associated with an increased risk of death from RCC (risk ratio, 1.38; 95% confidence interval, 1.03-1.84; P = 0.029). CONCLUSIONS: Both tumor cell and tumor vasculature B7-H3 expression convey important information to predict ccRCC outcomes. Collectively, our past and present studies pertaining to B7-H ligand expression indicate that ccRCC may use redundant mechanisms to compromise host antitumoral immunity. Future studies will focus on the effect of combined B7-H ligand expression in RCC.     

Variable expression of the interleukin-2 receptor alpha chain and MYC genes in lymphocytes from renal cell carcinoma patients treated with interleukin-2.

We have studied the expression of the interleukin-2 receptor alpha chain, c-MYC and L-MYC genes in lymphocytes obtained from four renal cell carcinoma patients undergoing an interleukin-2 clinical trial. Two of these patients exhibited stable disease after the interleukin-2 therapy and two exhibited progressive disease. Analysis of mRNA levels by dot blot hybridization indicated that changes in the expression of both the interleukin-2 receptor alpha chain and c-MYC genes were erratic and varied widely between patients. L-MYC expression was not observed in any sample. There appeared to be little correlation between the changes in gene expression and parameters such as thymidine incorporation, the proportion of CD25 positive cells present or cytotoxic activity. The situation in vivo therefore appears to be more complex than would be predicted from in vitro studies.     

Relationship between DNA ploidy, antigen expression and survival in renal cell carcinoma

Tumor ploidy and expression of renal differentiation antigens as recognized by a previously described series of monoclonal antibodies (MAbs) (RC2, RC3, RC4, RC38, RC69, RC154 and G250) were assessed in 48 renal-cell carcinomas. A positive association was found between aneuploidy and development of metastases. Aneuploid tumors showed a loss of antigen expression more frequently than diploid tumors. This difference was statistically significant for antigens recognized by RC3 and RC69. Loss of RC154 reactivity was associated with increased metastatic potential. Clinical stage was the most powerful single prognostic variable but ploidy and RC3 reactivity carried additional prognostic information.     

Expression of cyclooxygenase-2 in renal cell carcinoma: correlation with tumor cell proliferation, apoptosis, angiogenesis, expression of matrix metalloproteinase-2, and survival.

PURPOSE: Cyclooxygenase (COX)-2 plays an important role in tumor cell proliferation, resistance to apoptosis, angiogenesis, and invasion in various malignant tumors. However, the relationships between COX-2 expression and these biological processes, clinicopathological features, and survival rate in patients with renal cell carcinoma are not clear. EXPERIMENTAL DESIGN: Tumor sections surgically removed from 131 patients were examined for COX-2 expression by immunohistochemistry. We also examined Ki-67 labeling index, apoptotic index, microvessel density, and matrix metalloproteinase (MMP)-2 expression, and correlated COX-2 expression with various clinicopathological features and survival. RESULTS: Of 131 sections, 70 (53.4%) were positive for COX-2 expression. COX-2 expression was associated significantly with various clinicopathological features, and correlated with the Ki-67 labeling index, microvessel density, and MMP-2 expression (P < 0.01), but not with the apoptotic index (P = 0.054). COX-2 expression was also identified as an independent risk factor for large tumor size (>7 cm) in multivariate logistic regression model. COX proportional hazards analysis showed that distant metastasis and high T stage were independent prognostic factors [odds ratio (OR), 9.41; 95% confidence interval (CI), 2.16-41.11; P < 0.01 and OR, 5.19; 95% CI, 1.02-26.54; P = 0.048, respectively), whereas COX-2 expression was not (OR, 1.46; 95% CI, 0.24-9.00; P = 0.68). CONCLUSION: COX-2 expression in patients with renal cell carcinoma is associated with several clinicopathological factors, and appeared to play an important role in tumor cell proliferation and MMP-2 expression, but is not a significant prognostic factor.     

重组人源化白细胞介素2治疗转移性肾癌的临床研究

目的 观察重组人源化白细胞介素2皮下注射治疗转移性肾癌的疗效及安全性.方法 入组转移性肾癌患者41例,经重组人源化白细胞介素2皮下注射治疗2～4个周期,每个周期共5周.第1个周期:9百万单位(MIU)、每12 h 1次、第1～5天;第2～4个周期:9 MIU、每12 h1次、第1～2天,9 MIU、每天1次、第3～5天;停药1周后重复.2个周期后评价疗效,有效或稳定的患者继续2个周期治疗.结果 意向治疗人群(ITT)集中,完全缓解(CR)0例,部分缓解(PR)7例(17.1%),病情稳定(SD)19例(46.3%),疾病进展(PD)15例(36.6%),总有效率为17.1%[95%可信区间(CI)为5.6%～28.6%],疾病控制率为63.4%;中位疾病进展时间(TTP)为6个月,中位总生存时间(OS)为22.5个月,1年生存率为71.2%.符合方案人群(PP)集中,CR 0例,PR 7例(19.4%),SD16例(44.4%),PD 13例(36.1%),总有效率为19.4%(95%CI为6.5%～32.3%),疾病控制率为63.9%;中位TTP为6个月,中位OS未达到,1年生存率为66.7%.随访显示,治疗获益能够改善患者的长期生存.患者的不良反应以轻中度为主,包括疲乏感(100%)和发热(82.9%)等,3级以上不良反应少见.结论 重组人源化白细胞介素2治疗转移性肾癌在中国人中具有一定的疗效,且能延长患者的生存期,其不良反应大部分患者能够耐受.     

Linomide and interleukin-2 in patients with advanced renal cell carcinoma

Quinoline-3-carboxamide (Linomide) is a novel, synthetic immunomodulator acting via immunologic and non-immunologic mechanisms. It has shown efficacy against various malignancies, experimental autoimmune encephalomyelitis, and septic shock in animal models and has been investigated for clinical use in minimal residual myeloid leukemia with promising results. Interleukin-2 has shown considerable efficacy in palliative anti-tumor-treatment of advanced renal cell cancer, revealing remission rates of up to 40% in combination therapy regimens. Linomide is reported to exhibit synergistic effects with interleukin-2. Here we report on a clinical phase I/II study examining tolerance and efficacy of a combination therapy schedule of SQ interleukin-2 and PO Linomide in advanced renal cell cancer. Seventeen patients received 10 IU/m2 interleukin-2 per week for 8 weeks, resting interleukin-2 for another 8 weeks. In week 5 they started 5 mg Linomide daily, continued with 10 mg from week 7 to 16. No objective remissions were observed. Among 15 patients evaluable for response, 10 (66.7%) were progredient during the study. Three patients died during the observation period, including two not evaluable for response. Median survival was 4.0 months, median progression-free survival 2.5 months with a Kaplan-Meier estimate of 3.63 months. Fever, reduced general condition, nausea/vomiting, dyspnea, anorexia, chills and hypotension were the most common side effects, reaching WHO grade 3 in 6 and grade 4 in 2 cases. In summary, Linomide in combination with interleukin-2 provides no advantages in efficacy or toxicity over other therapy regimens employing interleukin-2.     

肾细胞癌中Egfl7表达与血管生成的相关性研究

目的:探讨肾细胞癌组织中Egfl7的表达对肾细胞癌血管生成的作用和意义.方法:应用SP 法对82 例肾癌及6例正常肾组织标本进行Egfl7和CD34 免疫组化染色,检测其Egfl7表达和CD34 标记的微血管密度(MVD)值,分析Egfl7表达和MVD之间,及其与肾癌临床病理之间的关系.结果:82 例肾癌病例中,57 例Egfl7表达阳性,总阳性率为69.5%.Egfl7表达与肾癌组织学分类、分期、分级和静脉浸润有关(P<0.05).Egfl7阳性表达和阴性表达组织中MVD 值分别为127.3±31.3和79.4±27.2, 差别有显著性(P＝0.001),且MVD 和Egfl7的表达呈正相关(r=0.875,P<0.05).结论:Egfl7可能在促进肾细胞癌血管生成中起重要作用.     

Targeting pulmonary metastases of renal cell carcinoma by inhalation of interleukin-2.

INTRODUCTION: Pulmonary metastases of renal cell carcinoma (RCC) are associated with poor prognosis. Inhalation therapy with interleukin-2 (IL-2) is thus an appealing method for palliation. This multicenter study summarizes the national experience of IL-2 inhalation in patients with lung metastases of RCC. PATIENTS AND METHODS: Forty patients (median, 66.5 years of age) with radiologically documented progressing pulmonary metastases were enrolled. All patients had to be able to comply with inhalation technique, and were not candidates for other treatment options. Twenty-eight patients were systemic treatment-na?ve. The protocol included three daily inhalations of IL-2 to a total dose of 18 MU. Treatment had to be continued until one of the following occurred: progression; a complete response; a life threatening toxicity; or patient refusal. Response was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) system. RESULTS: The disease-control rate reached 57.5%, with a partial response rate of 2.5% and a disease stabilization rate of 55%. Median time to progression was 8.7 months. The main side-effects were cough and weakness. CONCLUSIONS: Inhalation of IL-2 for the treatment of pulmonary metastases in RCC is feasible, tolerable and beneficial in controlling progressive disease for considerable periods of time. The definition of response of biological therapy may need to be re-assessed and modified: stable disease should be regarded as a favorable response.     

Osteopontin expression correlates with prognostic variables and survival in clear cell renal cell carcinoma

BACKGROUND AND OBJECTIVES: Osteopontin (OPN) is a phosphorylated glycoprotein with diverse functions including tumorigenesis and tumor cell metastasis. Recently, it has been detected in a growing number of human tumors, and assessed as a potential prognostic marker. The aim of this study was to analyze the expression of OPN in normal renal tissue and clear cell renal cell carcinomas (CRCCs), and to assess its prognostic significance. METHODS: The expression of OPN protein was immunohistochemically analyzed in 171 CRCCs and compared to usual clinicopathological parameters such as tumor size, nuclear grade, pathological stage, Ki-67 proliferation index, and cancer-specific survival. RESULTS: In normal renal parenchyma, the expression of OPN was seen in distal tubular epithelial cells, calcifications, and some stromal cells. The upregulation of OPN was observed in 61 CRCCs (35.7%) in the form of cytoplasmic granular staining of various intensities. Statistical analysis showed correlation of the OPN expression with tumor size (P < 0.001), Fuhrman nuclear grade (P < 0.001), pathological stage (P = 0.011), and Ki-67 proliferation index (P < 0.001). Moreover, patients with OPN-positive tumors had significantly worse prognosis in comparison to patients with tumors lacking OPN protein (P = 0.004). CONCLUSION: Our results suggest that overexpression of OPN is involved in the progression of CRCC.